Can you identify bee guts infected with Nosema?

Last week I needed to collect a fresh Nosema ceranae sample from a honey bee to use in an experiment.  We generally have a high prevalence of Nosema ceranae in the colonies here at the Bee Research Lab in Maryland, and a heavily infected individual is not too difficult to locate.  But, how do I tell if a bee is infected or not?  Some people say they can tell by looking at the midgut region of the bee's digestive tract whether or not they are infected by Nosema.  The midgut is the specific region of the honey bee gut that both species of Nosema (Nosema apis and Nosema ceranae) infect.

Do you think you can identify a Nosema infection by looking at the midgut?

Here's the test

• Adult workers were collected from an apparently healthy colony on July 3rd (that's right; no sign of dysentery, nosemosis, excessive bee poop on the porch, etc.) and brought to the lab.

• Bees were cold anesthetized on ice.

• Digestive tracts were then removed from 9 individual bees, shown below with both light (left) and dark (right) background.

Can you identify which of the 9 midguts shown are infected by Nosema ceranae?

 

Hints 

1.  At least one midgut shown is heavily infected with about 50 million Nosema spores.

2.  At least one midgut shown has no detectable Nosema spores.

Got your answer?  Give up???  Scroll down for the answers.

Here are light microscope images (400x) of the contents from each midgut shown above:

Only midgut #2 is infected with Nosema ceranae.  In some (#1, #4, #8, #9) large pollen grains are visible.  Some cellular debris (including what look to be lipid spheres) can be seen in the remaining images.  If you answered this correctly you were either a) lucky or b) know something I don't and were lucky.

What does this mean?

Gross examination of the midgut itself is not a reliable way to identify infected individuals.  I can never tell with any measure of confidence whether or not a midgut is infected, and I've looked at a lot of midguts!  It has been suggested that a field test can be used to diagnose Nosema infected midguts by visual inspection, looking for a light color and swelling.  This may be true for pure Nosema apis infections (with which I don't have experience), but is not true for the now most prevalent species N. ceranae and is also probably not true for mixed species infections.  As shown above, the lightest colored midguts (#5, #8, #9) were not infected and midguts equally large (#1, #7, #9) as the infected one (#2) were not infected.

How do I know this is Nosema ceranae?  

Because I extracted DNA from this sample and used a molecular diagnostic test called polymerase chain reaction (PCR) with empirically determined species-specific primers that can differentiate Nosema ceranae from Nosema apis. (See Table S1 below from R. Schwarz and J. Evans. 2013. Single and mixed-species trypanosome and microsporidia infections elicit distinct, ephemeral cellular and humoral immune responses in honey bees.  Developmental and Comparative Immunology 40, 300-310. http://dx.doi.org/10.1016/j.dci.2013.03.010 ).  Although N. ceranae spores are slightly smaller than N. apis, molecular diagnostics are the only way Nosema species identification can be reliably made in my opinion.